The invention described herein relates to a process for preparing transplantable tissue which has been cryopreserved and stored at cryopreservation temperatures. Success of certain tissue transplants relies in large measure upon the level of cell and tissue viability, which is defined as the ability of a cell or tissue to maintain itself and interact in a normal manner with its environment. With the steady increase in frequency of transplant surgery, techniques for tissue preservation, storage, thawing and cryoprotectant elution have become increasingly important for preserving tissue viability.
The invention described herein relates to a process for treating transplantable tissue after cryopreservation and thawing so as to elute (or dilute) the intracellular cryoprotectant which is frequently used. Intracellular cryoprotectants pose particular difficulties during transplant preparation due to temperature dependent chemical cytotoxicity. In addition, osmotic stress can be created due to hyperosmotic levels of solutes in the thawed cells.
Dilution techniques used to elute the cryoprotectant from cryopreserved tissues after thawing have traditionally used a multi-step procedure, seeking to minimize both the chemical cytotoxicity and the cellular osmotic shock, which in combination result in cell stress and potentially in cell death. Stepwise elution of the intracellular cryoprotectant minimizes the net diffusion of water into the cells, and permits the intracellular cryoprotectant to diffuse out. The multi-step approach tends to be time consuming and requires particular attention to details such as elution times, temperatures, etc. Since this relatively complex elution process is typically conducted in the operating room during a surgical procedure, it is advantageous to use the simplest, safest thawing and elution process with the least number of steps, thus permitting the operating room personnel to focus on the surgical procedure at hand.
The object of the present invention is to provide a one-step intracellular cryoprotectant elution method which reduces the cryoprotectant concentration within tissues to substantially non-toxic levels (to the constituent cells of the tissue) at which the tissue can be safely transplanted.
Another objective of the present invention is to provide a dilution process which requires a minimum number of steps.
Another objective of the present invention is to provide a process which requires no end point titration or determination.
Another objective of the present invention is to speed and simplify the elution process for the preparation of cryopreserved tissue in order to facilitate transplant surgery. These and other objectives will be apparent to those of ordinary skill in the art from the teachings herein.